The Notch signaling pathway is necessary for vascular development, and controls differentiation and maturity of both endothelial cells and smooth muscle cells. Activation of Notch transcriptionally regulates target genes such as hairy/enhancer of split (HES), HES-related transcription factor (HRT), and others. Our goal is to understand the mechanisms by which Notch and HRT signaling regulate smooth muscle cell phenotype. Although the Hes/HRT transcriptional regulators are considered Notch effector molecules that mediate Notch activity on transcriptional targets, this is not the case for smooth muscle marker expression in human primary cells. HRT expression negatively regulates Notch-induced cyclinD3, smooth muscle alpha-actin, calponin, and smooth muscle-myosin heavy chain proteins. While some of this activity may be due to HRT interaction with CBF-1, these pathways are not characterized. Our objective is to understand the interaction of Notch/HRT signaling as a balance between signals leading to smooth muscle differentiated phenotype, versus a transitional phenotype in which characteristics of differentiation are decreased to allow for cell proliferation and remodeling. The role of the endothelium in these processes will also be addressed. Overall Hypothesis: Notch signaling selectively activates gene targets that lead to the mature differentiated phenotype of human primary smooth muscle cells. Notch activity, in turn, is antagonized by HRT activity, which is a natural "off switch" for Notch activity in the smooth muscle cell. Specific Aim 1: To identify the mechanisms by which Notch signaling induces the expression of smooth muscle differentiation gene targets (cyclinD3 and SMA), and the mechanisms by which HRT antagonizes Notch function. Specific Aim 2: To analyze the heterotypic interactions mediated by Notch signaling from the endothelial cell to the smooth muscle cell, using a co-culture model. Specific Aim 3: To characterize smooth muscle development in vivo in transgenic models of Notch/HRT signaling in smooth muscle, focusing on the yolk sac vasculature. The accomplishment of these aims will further our understanding of regulators of smooth muscle behavior during vascular disease. PUBLIC HEALTH RELEVANCE Cardiovascular diseases comprise a major health problem that at the cellular level involve defective function of cells in the blood vessel. Genetic mutations in the Notch pathway are present in some patients with diseases that lead to stroke and blood vessel disease. Our goal is to study this pathway at the genetic, molecular, and cellular level, to expand our knowledge in developing treatments for cardiovascular disease.